Priming of the oxidative burst in rice and wheat cell cultures by ulvan,a polysaccharide from green macroalgae,and enhanced resistance against powdery mildew in wheat and barley plants

The capacity of ulvan, a sulphated heteropolysaccharide, to prime the chitin‐ and chitosan‐elicited oxidative burst in wheat and rice cells was tested. Gas‐liquid chromatography showed that ulvan was composed of rhamnose, xylose, glucuronic acid, glucose and galactose. It contained very low amounts of proteins and ca. 19% sulphate groups. The polymer did not elicit the production of hydrogen peroxide in suspension‐cultured wheat or rice cells. Furthermore, in both cell cultures, the simultaneous addition of ulvan and chitin hexamer or chitosan polymer did not significantly alter the intensity of the oxidative burst caused by the elicitors alone. However, pretreatment of wheat cells with ulvan increased the chitin‐elicited oxidative burst about five‐ to sixfold, and that elicited by chitosan about twofold. In rice cells, the production of H₂O₂ elicited by chitin or chitosan was strongly primed by pretreatment with the same concentration of ulvan, increasing the burst triggered by the elicitors alone by 150 and 80 times, respectively. Pretreatment of whole plants with ulvan significantly reduced the symptom severity of Blumeria graminis infection, by 45% in wheat and by 80% in barley. Thus, the priming activity of ulvan on the oxidative burst correlates with a decrease of disease symptoms in infected plants. This is apparently the first report on priming activity of a natural algal polysaccharide.     

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.     

Induction of peroxidase, scopoletin, phenolic compounds and resistance in Hevea brasiliensis by elicitin and a novel protein elicitor purified from Phytophthora palmivora

Elicitin and a new protein 75 kDa elicitor were purified from the culture filtrate of Phytophthora palmivora, a pathogen of Hevea brasiliensis (rubber plant). Elicitin was obtained by using a one step of DEAE cellulose chromatography and the new elicitor was obtained by two steps of chromatography: a DEAE cellulose column followed by a hydrophobic column. Both elicitors were stable to heat and a wide range of pH values, but were sensitive to ProteaseK. Both elicitors induced scopoletin, peroxidase isozymes (with substrate o-dianisidine and scopoletin) and total phenolic compounds in cell suspension of H. brasiliensis with similar kinetics. In addition, both elicitors induced peroxidase enzyme (o-dianisidine), total phenolic compounds and enhanced local resistance against P. palmivora on young rubber tree seedlings. However, the increase of peroxidase enzyme and total phenolic compounds in rubber tree seedlings was different from those in cell suspension. Furthermore, during the expression of local resistance the zoospore of P. palmivora induced the peroxidase enzyme (o-dianisidine) more rapidly and with higher level than the control plants. H. brasiliensis is more responsive to the new elicitor than elicitin in triggering defense responses. That is the new elicitor was active at a concentration lower than those required for elicitin, about a 30-fold decrease for activation defense responses in cell suspension. For induction of peroxidase enzyme (o-dianisidine), phenolic compounds and local resistance of rubber plants against P. palmivora, the 75 kDa protein was active at about a 2-fold lower concentration when compared to elicitin.     

Induction of a viable but not culturable (VBNC) state in some Pseudomonas syringae pathovars upon exposure to oxidation of an apoplastic phenolic,acetosyringone

Acetosyringone is a phenolic metabolite often found in plant apoplasts. Its oxidation by hydrogen peroxide and peroxidase results in a prolonged increase in the redox potential of the reaction mixture, similar to redox increases observed in tobacco suspension cells upon treatment with incompatible bacteria. Since high redox potentials, being oxidative, are generally detrimental to bacteria, the effect of acetosyringone oxidation on bacterial viability was examined. Pseudomonas syringae pv. syringae was added to reaction mixtures containing acetosyringone, hydrogen peroxide and peroxidase and samples were removed to determine viability by dilution plating. Initial studies were done with low bacterial concentrations, 10⁵ CFU ml⁻¹, to ensure that scavenging of H₂O₂ was negligible and did not interfere with the reaction mixture. No colonies were formed by bacteria that had been added to reaction mixtures with acetosyringone ranging from 25 to 100 μΜ. Examination of the bacteria by microscopy and flow cytometry, using fluorescent stains that indicate bacterial membrane integrity, suggested that these bacteria had maintained their membrane integrity. In addition they were able to respire based on oxygen uptake. When bacteria were added to on-going reaction mixtures at a time point after the prolonged redox response, the CFU ml⁻¹ increased indicating that a stable reaction product was not responsible for the non-culturability bioactive effect. Other bacterial isolates, P. s. pv. tabaci and Pseudomonas fluorescens, were less susceptible to the bioactive effect of the acetosyringone oxidation. Other phenolics were tested and had lesser degrees of bioactivity and in some cases reduced the bioactivity of acetosyringone oxidation. The ‘viable but non-culturable’ (VBNC) state of the bacteria in this study is compared to that described for other medical and plant pathogens.     

Metalaxyl-induced changes in the antioxidant metabolism of Solanum nigrum L. suspension cells

The antioxidant responses of Solanum nigrum L. cell suspension cultures to metalaxyl exposure were investigated. An increase in lipid peroxidation and hydrogen peroxide content, for both concentrations tested (20 mg L⁻¹; 40 mg L⁻¹) revealed the response of oxidative metabolism of cell suspensions to metalaxyl. Superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) activities increased, particularly in the highest concentration of metalaxyl used. An analysis by non-denaturing polyacrylamide gel (PAGE) followed by staining for enzyme activity, revealed seven SOD isoenzymes, two CAT isoenzymes, and nine APX isoenzymes. Metalaxyl levels were quantified in the culture medium and results suggest that suspension cells were able to accumulate and/or degrade the fungicide five hours after exposure. SOD, CAT and APX isoenzymes were differently affected by the metalaxyl treatment. Results suggest that the higher concentration of metalaxyl induced oxidative stress to cell suspension cultures of S. nigrum.     

Colonization of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots by <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> promotes growth and enhances systemic disease resistance through jasmonic acid/ethylene and salicylic acid pathways

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.     

Characterization of Pectolytic Erwinias as Highly Sophisticated Pathogens of plants

Erwinia carotovora and Erwinia chrysanthemi are the two most important soft rotting bacteria of commercially-grown plants. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribsomal gene cluster. Subpopulations grouped into biovars, pathovars, or subspecies associated with various hosts and in different geographic regions suggest specialization in host preference and/or survival in diverse environments. Previous characterization of the pectolytic erwinias as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. The presence of an entire hrp gene cluster and associated type III secretion system, and global regulators which regulate virulence determinants such as exoenzyme production and motility, attest to a highly specialized pathogen. The fact that production of extracellular plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum-sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness.     

Histochemical and chemical evidence for lignin accumulation during the expression of resistance to leaf rust fungi in wheat

Levels of individual phenolic acids were examined in primary leaves of wheat (Triticum aestivum) after inoculation with avirulent and virulent strains of the leaf rust fungus (Puccinia recondita f. sp. tritici) at stages when previous work had shown fungal and host cells to be affected by expression of the Lr20 or Lr28 alleles for resistance. The predominant phenolic acid, ferulic acid, as well as p-coumaric and syringic acids were detected in primary leaves in both unbound and bound forms. They were not detected in germinating urediniospores of either rust strain. Levels of unbound phenolic acids changed little in response to infection. In Lr28-bearing leaves inoculated with an avirulent strain, increased concentrations of bound phenolic acids and three other unidentified compounds were observed about 4 h after many single or small groups of cells had undergone hypersensitive collapse. In an Lr20-bearing cultivar, levels of bound phenolic acids fell in leaves inoculated with either a virulent or avirulent rust strain. Coniferyl aldehyde and coniferyl alcohol were not detected in healthy or inoculated leaves of either wheat cultivar. Attempts to affect expression of resistance by application of inhibitors of phenylalanine ammonia-lyase were not successful and both wheat cultivars remained resistant to avirulent rust strains. The bound phenolic acids which accumulate in cells undergoing a hypersensitive response may play a role in resistance of Lr28-bearing wheat to the leaf rust fungus.     

白叶枯病菌与非亲和水稻IRBB4悬浮细胞互作中蛋白类激发子的纯化和鉴定

 本研究对水稻白叶枯病菌与水稻悬浮细胞非亲和互作中蛋白类激发子进行了分离纯化和鉴定.白叶枯病菌JXOV与水稻IRBB4和IR24悬浮细胞互作36 h后的上清液,经Q-Sepharose阴离子交换层析柱分离,对分离的各组分进行抗病性诱导测定,结果表明JXOV与IRBB4非亲和互作的上清液中存在蛋白类激发子.有活性的蛋白组分经阴离子交换层析柱Mono-Q进一步纯化后,SDS-PAGE分析鉴定出2个具激发活性的蛋白,其分子量分别为17.2 kD和49.2 kD,等电点分别为5.8和6.2.利用上述激发子处理水稻能减少病斑长度并诱导水稻防卫酶活性的增加.     

The oligopeptide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactions in potato

The Phytophthora-derived oligopeptide elicitor, Pep-13, originally identified as an inducer of plant defense in the nonhost–pathogen interaction of parsley and Phytophthora sojae, triggers defense responses in potato. In cultured potato cells, Pep-13 treatment results in an oxidative burst and activation of defense genes. Infiltration of Pep-13 into leaves of potato plants induces the accumulation of hydrogen peroxide, defense gene expression and the accumulation of jasmonic and salicylic acids. Derivatives of Pep-13 show similar elicitor activity in parsley and potato, suggesting a receptor-mediated induction of defense response in potato similar to that observed in parsley. However, unlike in parsley, infiltration of Pep-13 into leaves leads to the development of hypersensitive response-like cell death in potato. Interestingly, Pep-13-induced necrosis formation, hydrogen peroxide formation and accumulation of jasmonic acid, but not activation of a subset of defense genes, is dependent on salicylic acid, as shown by infiltration of Pep-13 into leaves of potato plants unable to accumulate salicylic acid. Thus, in a host plant of Phytophthora infestans, Pep-13 is able to elicit salicylic acid-dependent and -independent defense responses.     

Functional imaging of biophoton responses of plants to fungal infection

Each living cell of a plant produces photons in certain conditions. Under normal physiological conditions, cell photon emission is stationary and minimal. Disturbance in the oxidative homeostasis by biotic stress is manifested by increased ‘biophoton’ production. Such biophoton responses of plants may be used as an integral indicator of the degree of oxidative homeostasis misbalance. Our results demonstrate that biophoton generation has been much higher in a resistant potato variety than in a susceptible one till 10 h after Phytophthora infestans inoculation. In contrast, ultra-weak luminescence from detached susceptible potato and moderately resistant pelargonium leaves increased from 1–4 to 4–5 days after inoculation with Phytophthora infestans or Botrytis cinerea, respectively. Pre-treatment of susceptible potato leaves with a defence inducer, arachidonic acid, resulted in a transient burst of light in response to P. infestans lasting for 30–45 h post inoculation (hpi). This study presents the potential adaptation of functional imaging of ultra-weak luminescence to monitor time-dependent free radical processes during disease development and its application to draw conclusions on plant resistance to pathogens of different lifestyle. Moreover, it has been shown that imaging of temporal biophoton generation from potato leaves treated with arachidonic acid might be a helpful marker in mapping oxidative changes leading to systemic acquired resistance (SAR).     

Apoplastic redox metabolism: Synergistic phenolic oxidation and a novel oxidative burst

The plant apoplast is an important mediator of communication between the cell cytoplasm and its surroundings. Plant cell suspensions offer a convenient model system to gain insight into apoplastic physiology. Here, we describe a novel phenomenon that took place when two naturally occurring phenolics were added together to either soybean or tobacco cell suspensions. Acetosyringone (AS) and/or hydroxyacetophenone (HAP), phenolics found in the extracellular/apoplast of tobacco cells, were added to soybean or tobacco cell suspensions undergoing an oxidative burst. Individually, AS appeared to be utilized as a typical peroxidase substrate to scavenge hydrogen peroxide, while HAP was utilized at a much lower rate. However, when added together the rate of utilization of both phenolics increased and surprisingly resulted in the production of hydrogen peroxide. We have further characterized this novel phenomenon in suspension cells. This study demonstrates that certain phenolics in plants can cause co-oxidation which, as in animals, could alter the structure and bioactivity of surrounding phenolics.     

Overexpression of StRbohA in Arabidopsis thaliana enhances defence responses against Verticillium dahliae

Plant NADPH oxidases are key regulators of plant–microbe interactions and reactive oxygen species (ROS) are essential to plant defences against pathogens. A significant part in the role played by ROS has been ascribed to plant respiratory burst oxidase homologs (RBOHs). In potato (Solanum tuberosum), where RBOHs were previously shown to be involved in wound-induced oxidative burst, we assessed their expression after inoculation with Verticillium dahliae Kleb. and showed that StRbohA was the only homolog to be differentially induced in potato in response to inoculation. In order to investigate the potential role of this gene in plant protection against wilt diseases, we used Agrobacterium-mediated transformation of Arabidopsis to assess the effects of its overexpression on plant responses to V. dahliae. After inoculation with this pathogen, the transformed Arabidopsis line overexpressing StRbohA showed lower disease severity (percent damaged leaf area and vascular discoloration) as compared to the wild type. It also had higher ROS production and more cell death caused by hydrogen peroxide (H₂O₂), compared to the wild type. Suberization of root cells was also more pronounced in the line overexpressing StRbohA, and supports a possible role for StRBOHA in plant resistance to V. dahliae. Together, these findings indicate that overexpressed StRbohA in Arabidopsis enhances the ROS-mediated defence mechanisms against V. dahliae and can be a potential tool to improve plant resistance to this and other soilborne pathogens that cause wilts in economically important crops.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Riboflavin activates defense responses in tobacco and induces resistance against Phytophthora parasitica and Ralstonia solanacearum

The effects of riboflavin on defense responses and secondary metabolism in tobacco (Nicotiana tabacum cv. NC89) cell suspensions and the effects of protecting tobacco seedlings against Phytophthora parasitica var. nicotianae and Ralstonia solanacearum were investigated. Defense responses elicited by riboflavin in tobacco cells included an oxidative burst, alkalinization of the extracellular medium, expression of 4 defense-related genes with different kinetics and intensities, and accumulation of 2 total phenolic compounds, scopoletin and lignin. When applied to tobacco plants challenged by P. parasitica and R. solanacearum, riboflavin treatment resulted in 47.9% and 48.0% protection, respectively. These results suggest that riboflavin can both induce a series of defense responses and secondary metabolism in cell suspensions and protect tobacco against P. parasitica and R. solanacearum.     

Isobutyl amides--potent compounds for controlling Diatraea saccharalis

BACKGROUND: A dichloromethane‐methanol extract of the seeds of Piper tuberculatum Jacq. (Piperaceae) and two isobutyl amides, 4,5‐dihydropiperlonguminine (1) and pellitorine (2), which were isolated by chromatographic methods, were assayed for their lethality against the sugarcane borer Diatraea saccharalis F. (Lepidoptera: Pyralidae). RESULTS: Bioassays were carried out with fourth‐instar caterpillars through topical application of test solutions to the dorsal surface of the prothorax, and dose–response correlations were determined. Significant insect mortalities were observed 24, 48 and 72 h after treatment at concentrations of ≥ 100 µg insect^?1. The LD₅₀ and LD₉₀ values for compound 1 were 92.83 and 176.50 µg insect^?1, and for compound 2 they were 91.19 and 184.56 µg insect^?1. CONCLUSION: According to the LD₅₀ and LD₉₀ for compounds 1 and 2, it can be inferred that the values reflect an acute lethal response to both compounds, based on interaction(s) of the toxicants with a primary target or series of targets. Thus, the amides were demonstrated to have potential value in the control of the sugarcane borer. Copyright © 2008 Society of Chemical Industry     

Signal Interactions in Induced Resistance to Pathogens and Insect Herbivores

Plants are often simultaneously challenged by pathogens and insects capable of triggering an array of responses that may be beneficial or detrimental to the plant. The efficacy of resistance mechanisms can be strongly influenced by the mix of signals generated by biotic stress as well as abiotic stress such as drought, nutrient limitation or high soil salinity. An understanding of their biochemical nature, and knowledge of the specificity and compatibility of the signaling systems that regulate the expression of inducible responses could optimize the utilization of these responses in crop protection. Signaling conflicts and synergies occur during a plant's response to pathogens and insect herbivores, and much of the research on defense signaling has focused on salicylate- and jasmonate-mediated responses. We will review our results using tomato (Lycopersicon esculentum) in greenhouse and field studies that illustrate a trade-off between salicylate- and jasmonate-mediated signaling, and discuss research on strategies to minimize the trade-off that can occur following the application of chemical elicitors of resistance. In addition, there is evidence of another signaling system that mediates endogenous levels of ceramide in the plant. This signal is associated with programmed cell death and protection of tomato against the fungal pathogen Alternaria alternata f. sp. lycopersici.     

Susceptibility of Broccoli Cultivars to Bacterial Head Rot: In Vitro Screening and the Role of Head Morphology in Resistance

Head rot is a major disease of broccoli caused by the soft rot pathogens Pseudomonas fluorescens and Erwinia carotovora. Two in vitro pathogenicity tests were evaluated as a methods to identify broccoli cultivars susceptible or resistant to bacterial head rot. One test used mature heads excised from the plant and inoculated with squares of cotton lint which had been soaked in a bacterial suspension. The other test involved stab-inoculating axenically grown seedlings. With the excised head test, susceptible cultivars showed a black soft rot, whilst less susceptible or moderately resistant cultivars showed only watersoaking, or browning and slight softening of the tissue. No cultivar was completely resistant. Ten cultivars were tested, and their susceptibility ratings corresponded with previously recorded field data, with one exception. This laboratory test could be used to screen for susceptibility to head rot in broccoli breeding programmes. The seedling test distinguished differences in aggressiveness among bacterial isolates but not cultivar susceptibility. Increasing head size correlated negatively with disease resistance. Head shape, i.e. cultivars which showed a domed shape rather than a flat shape, was positively correlated with disease resistance. Thus small domed heads are more resistant to head rot than large flat heads. Other morphological characteristics, viz. floret prominence and number, and sepal stomatal number were not correlated with host resistance.     

Cytokinin induces bacterial pathogen resistance in tomato

Phytohormones are involved in the regulation of plant responses to biotic stress. How a limited number of hormones differentially regulate defence responses and influence the outcome of plant–biotic interactions is not fully understood. In recent years, cytokinin (CK) was shown to induce plant resistance against several pathogens. In the present study, we investigated the effect of CK in inducing tomato resistance against the hemibiotrophic pathogenic bacteria Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv. tomato (Pst). We demonstrate that CK enhances tomato resistance to Xcv and Pst through a process that relies on salicylic acid and ethylene signalling. CK did not directly affect the growth or biofilm formation ability of these pathogens in vitro. Overall, our work provides insight into the underlying mechanisms of CK-induced immune responses against bacterial pathogens in tomato.     

Oxygen metabolism in plant cell culture/bacteria interactions: role of bacterial concentration and H2O2-scavenging in survival under biological and artificial oxidative stress

Preliminary studies with plant cell suspensions indicated that high concentrations of bacterial inoculum can significantly reduce the H₂O₂levels detected immediately following inoculation. Further investigation suggested that bacterial concentration is an important factor in H₂O₂-scavenging, and is often overlooked in both plant/pathogen interactions as well asin vitrosurvival studies.In vitrostudies withPseudomonasspp. characterized the relationship between H₂O₂-scavenging and culturability of bacteria. Because H₂O₂permeates membranes rapidly, the intracellular scavenging activity of the bacteria could be detected as a decrease in the external H₂O₂concentration. Therefore, the effective scavenging activity of a given suspension of bacteria was proportional to the bacterial concentration. The culturability of populations of bacteria exposed to H₂O₂concentrations which are intolerable to individual cells (≥100 μm) was also dependent on bacterial concentration. Studies with a catalase-deficient mutant ofP. putidaindicated that catalase, due to its high K_m, had little effect on scavenging H₂O₂at the lower concentrations of H₂O₂(<100 μm) that are likely to be encountered in suspension cells and other plant tissues. Using high concentrations of bacteria (1×10⁹cfu ml^-1), there was little difference between the catalase-deficient mutant and various isolates ofPseudomonasspp. in the reduction of H₂O₂levels in suspension cells. The studies indicated that an additional scavenging system other than catalase exists in bacteria and this may play an important role in scavenging H₂O₂during plant/bacteria interactions.